Inhibition of αIIbß3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the ßI Domain of ß3.

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbß3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the ß-ribbon extending from the ß-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbß3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the ß-ribbon by forming a clasp restraining the ß3 hybrid and ßI domains in a closed conformation. The involvement of species-specific residues of the ß3 hybrid domain (E356 and K384) and the ß1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbß3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb ß-ribbon in preventing integrin αIIbß3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

Receptor protein tyrosine phosphatase beta/zeta is a functional binding partner for vascular endothelial growth factor.

BACKGROUND: Receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN). RPTPß/ζ interacts with ανß3 on the cell surface and upon binding of PTN leads to c-Src dephosphorylation at Tyr530, ß3 Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated ß3 Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF165) stimulation of endothelial cells and is essential for VEGF receptor type 2 (VEGFR2) - ανß3 integrin association and subsequent signaling. In the present work, we studied whether RPTPß/ζ mediates angiogenic actions of VEGF. METHODS: Human umbilical vein endothelial, human glioma U87MG and stably transfected Chinese hamster ovary cells expressing different ß3 subunits were used. Protein-protein interactions were studied by a combination of immunoprecipitation/Western blot, immunofluorescence and proximity ligation assays, properly quantified as needed. RPTPß/ζ expression was down-regulated using small interference RNA technology. Migration assays were performed in 24-well microchemotaxis chambers, using uncoated polycarbonate membranes with 8 µm pores. RESULTS: RPTPß/ζ mediates VEGF165-induced c-Src-dependent ß3 Tyr773 phosphorylation, which is required for VEGFR2-ανß3 interaction and the downstream activation of phosphatidylinositol 3-kinase (PI3K) and cell surface NCL localization. RPTPß/ζ directly interacts with VEGF165, and this interaction is not affected by bevacizumab, while it is interrupted by both CS-E and PTN. Down-regulation of RPTPß/ζ by siRNA or administration of exogenous CS-E abolishes VEGF165-induced endothelial cell migration, while PTN inhibits the migratory effect of VEGF165 to the levels of its own effect. CONCLUSIONS: These data identify RPTPß/ζ as a cell membrane binding partner for VEGF that regulates angiogenic functions of endothelial cells and suggest that it warrants further validation as a potential target for development of additive or alternative anti-VEGF therapies.

Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species.

Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and integrin alpha v beta 3 (ανß3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPß/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN-ανß3 interaction abolished PTN-induced ROS generation, suggesting that ανß3 is also involved. The latter was confirmed in CHO cells that do not express ß3 or over-express wild-type ß3 or mutant ß3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type ß3 but not in cells not expressing or expressing mutant ß3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανß3 and RPTPß/ζ and activation of c-src, PI3K and ERK1/2 kinases.

Interplay between αvß3 integrin and nucleolin regulates human endothelial and glioma cell migration.

The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because α(v)ß(3) integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and α(v)ß(3) by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing α(v)ß(3) and depended on the phosphorylation of ß(3) at Tyr(773) through receptor protein-tyrosine phosphatase ß/ζ (RPTPß/ζ) and c-Src activation. Downstream of α(v)ß(3,) PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both α(v)ß(3) and RPTPß/ζ. Positive correlation of cell surface NCL and α(v)ß(3) expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing α(v)ß(3). Collectively, these data suggest that both expression and ß(3) integrin phosphorylation at Tyr(773) determine the cell surface localization of NCL downstream of the RPTPß/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.

Redox control of the survival of healthy and diseased cells.

Abstract Cellular redox homeostasis is the first line of defense against diverse stimuli and is crucial for various biological processes. Reactive oxygen species (ROS), byproducts of numerous cellular events, may serve in turn as signaling molecules to regulate cellular processes such as proliferation, differentiation, and apoptosis. However, when overproduced ROS fail to be scavenged by the antioxidant system, they may damage cellular components, giving rise to senescent, degenerative, or fatal lesions in cells. Accordingly, this review not only covers general mechanisms of ROS production under different conditions, but also focuses on various types of ROS-involved diseases, including atherosclerosis, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases, and cancer. In addition, potentially therapeutic agents and approaches are reviewed in a relatively comprehensive manner. However, due to the complexity of ROS and their cellular impacts, we believe that the goal to design more effective approaches or agents may require a better understanding of mechanisms of ROS production, particularly their multifaceted impacts in disease at biochemical, molecular, genetic, and epigenetic levels. Thus, it requires additional tools of omics in systems biology to achieve such a goal. Antioxid. Redox Signal. 15, 2867-2908.

PML/RARalpha fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association.

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

Recent advances in the understanding of the molecular mechanisms regulating platelet integrin αIIbß3 activation.

Integrins are allosteric cell adhesion receptors that cycle from a low to a high affinity ligand binding state, a complex process of receptor activation that is of particular importance in blood cells such as platelets or leukocytes. Here we highlight recent progress in the understanding of the molecular pathways that regulate integrin activation in platelets and leukocytes, with a special focus on the structural changes in platelet integrin αIIbß3 brought about by key intracellular proteins, namely talin and kindlins, that are of crucial importance in the regulation of integrin function. Evidence that the small GTPase Rap1 and its guanine exchange factor CalDAG-GEF1, together with RIAM, a Rap1GTP adaptor protein, promote the interaction of talin with the integrin ß subunit, has greatly contributed to fill the gap in our understanding of the signaling pathway from G-coupled agonist receptors and their phospholipase C-dependant second messengers, to integrin activation. Studies of patients with the rare blood cell disorder LAD-III have contributed to the identification of kindlins as new co-regulators of the talin-dependent integrin activation process in platelets and leukocytes, underlining the relevance for the in-depth investigation of patients with rare genetic blood cell disorders.

The novel S527F mutation in the integrin beta3 chain induces a high affinity alphaIIbbeta3 receptor by hindering adoption of the bent conformation.

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alphaIIbbeta3 in a patient with an ill defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated alphaIIbbeta3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alphaIIbbeta3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the alphaIIbbeta3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe(527) in beta3 and the calf-1 domain in alphaIIb and by decreased flexibility between I-EGF2 and I-EGF3.

Integrin alpha(v)beta(3) is a pleiotrophin receptor required for pleiotrophin-induced endothelial cell migration through receptor protein tyrosine phosphatase beta/zeta.

We have previously shown that the angiogenic growth factor pleiotrophin (PTN) induces migration of endothelial cells through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). In this study, we show that a monoclonal antibody against alpha(nu)beta(3) but not alpha(5)beta(1) integrin abolished PTN-induced human endothelial cell migration in a concentration-dependent manner. Integrin alpha(nu)beta(3) was found to directly interact with PTN in an RGD-independent manner, whereas a synthetic peptide corresponding to the specificity loop of the beta(3) integrin extracellular domain ((177)CYDMKTTC(184)) inhibited PTN-alpha(nu)beta(3) interaction and totally abolished PTN-induced endothelial cell migration. Interestingly, alpha(nu)beta(3) was also found to directly interact with RPTPbeta/zeta, and PTN-induced Y773 phosphorylation of beta(3) integrin was dependent on both RPTPbeta/zeta and the downstream c-src kinase activation. Midkine was found to interact with RPTPbeta/zeta, but not with alpha(nu)beta(3), and caused a small but statistically significant decrease in cell migration. In the same line, PTN decreased migration of different glioma cell lines that express RPTPbeta/zeta but do not express alpha(nu)beta(3), while it stimulated migration of U87MG cells that express alpha(nu)beta(3) on their cell membrane. Overexpression or down-regulation of beta(3) stimulated or abolished, respectively, the effect of PTN on cell migration. Collectively, these data suggest that alpha(nu)beta(3) is a key molecule that determines the stimulatory or inhibitory effect of PTN on cell migration.

Testicular cancer in Luxembourg: incidence and outcome in relation to the different histo-pathological types (1980-2004).

BACKGROUND: Increasing incidence rates of testicular cancer have been reported worldwide over the last three decades. Trends over time in the incidence rates of germ cell tumours (GCTs) in Luxembourg (Western Europe) and the outcome, both in relation to the different histological types, were analysed. METHODS: The population-based files of the Morphologic Tumour Registry collecting at a nation-wide level all testicular cancers diagnosed between 1980 and 2004 in the central department of pathology in Luxembourg were retrospectively reviewed. In addition, the presence of concomitant malignant diseases was investigated. RESULTS: 397 patients with GCT were evaluated. The mean age was 33.7 years (range: 1-72). Most of the patients (58.7%) were between 15 and 34 years of age. The age-standardized incidence rates rose from 4.5 per 10(5) (1980-1984) to 7.7 per 10(5) (2000-2004). Out of 275 (69.3%) pure GCTs, 218 seminomas, 48 embryonal carcinomas, 4 yolk sac tumours, 4 malignant teratomas and 1 choriocarcinoma were identified. 30.7% GCTs were of mixed type with 17 different histological variants. 5.8% of the patients had metachronous concomitant cancers, 2% bilateral GCTs and 3.8% non-testicular neoplasms. In all histological categories, with the exception of the pure seminomas, prognosis was determined within the 24 months following diagnosis; pure seminomas need long time follow-up. Ten-year observed survival rates exceeded mostly 90%. Pure embryonal carcinomas had the worst prognosis with a 10-year observed survival rate of 87.1 +/- 12%. CONCLUSIONS: Testicular germ cell tumours are rare, highly curable neoplasms that generally occur in young patients. In all histological categories, except for pure seminomas, prognosis was determined within the 24 months after diagnosis. Despite better observed survival rates, patients with pure seminomas, without or with metachronous concomitant non-testicular malignancies, need long time followup strategy.

The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.

The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

The talin rod IBS2 alpha-helix interacts with the beta3 integrin cytoplasmic tail membrane-proximal helix by establishing charge complementary salt bridges.

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin beta subunits to the cytoskeleton ( Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280-17288 ). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3-talin rod interaction relies on a helix-helix association between alpha-helix 50 of the talin rod domain and the membrane-proximal alpha-helix of the beta3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation.

RGT, a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase.

Mutational analysis has established that the cytoplasmic tail of the integrin beta 3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin beta 3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin beta 3 and dose-dependently inhibited the purified recombinant beta 3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the beta 3 cytoplasmic tyrosines, Y(747) and Y(759), was inhibited by myr-RGT. These data indicate an important role for beta 3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with beta 3 and selective blockade of integrin alpha IIb beta 3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia.

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.

The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin beta subunits to the cytoskeleton.

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1(-/-) cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.

Complete loss of PTEN expression as a possible early prognostic marker for prostate cancer metastasis.

The EGF/IGF growth factors are potent mitogens that regulate cell proliferation and cell survival and are involved in prostate cancer development. Using laser microdissection technology and real-time PCR, together with immunohistochemistry, we have explored the growth factor and integrin dependent PI3-kinase/PTEN/Akt signalling pathway in prostate cell lines and tumour samples by analysing EGF-R, IGF1-R, ILK, beta3 integrin, PTEN and p-Akt protein expression. We provide evidence that loss of PTEN expression rather than upregulated EGF/IGF1 receptor expression was responsible for increased p-Akt in neoplastic prostate cells. We therefore compared PTEN expression in patient biopsies at first time diagnosis recruited prospectively (Study I, 112 patients) and patients with confirmed metastasis recruited retrospectively from the Luxembourg cancer registry (Study II, 42 patients). In Study I, loss of PTEN expression at first time diagnosis was found in 26 of 112 patients (23%). In Study II, 25 of the 42 patients (59%) with lymph node metastasis had complete loss of PTEN expression in both the neoplastic glands of the prostate and the invasive prostate cancer cells in the lymph node, and of these 13 (52%) exhibited already loss of PTEN expression at first diagnosis. These findings demonstrate that loss of PTEN expression is an important factor in progression towards metastatic disease and could potentially serve as an early prognostic marker for prostate cancer metastasis.

Effect of PEX, a noncatalytic metalloproteinase fragment with integrin-binding activity, on experimental Chlamydophila pneumoniae infection.

Chlamydophila pneumoniae is a pathogen that is involved in acute and chronic respiratory infections and that is associated with asthma and coronary artery diseases. In this study, we evaluated the effects of PEX, a noncatalytic metalloproteinase fragment with integrin-binding activity, against experimental infections caused by C. pneumoniae. Moreover, we investigated the relationships between C. pneumoniae and alpha(v)beta(3) integrin functions in order to explain the possible mechanism of action of PEX both in vitro and in vivo. For the in vitro experiments, HeLa cells were infected with C. pneumoniae and treated with either PEX or azithromycin. The results obtained with PEX were not significantly different (P > 0.05) from those achieved with azithromycin. Similar results were also obtained in a lung infection model. Male C57BL/J6 mice inoculated intranasally with 10(6) inclusion-forming units of C. pneumoniae were treated with either PEX or azithromycin plus rifampin. Infected mice treated with PEX showed a marked decrease in C. pneumoniae counts versus those for the controls; this finding did not differ significantly (P > 0.05) from the results observed for the antibiotic-treated group. Integrin alpha(v)beta(3) plays an important role in C. pneumoniae infection. Blockage of integrin activation led to a significant inhibition of C. pneumoniae infection in HeLa cells. Moreover, CHO(DHFR) alpha(v)beta(3)-expressing cells were significantly (P < 0.001) more susceptible to C. pneumoniae infection than CHO(DHFR) cells. These results offer new perspectives on the treatment of C. pneumoniae infection and indicate that alpha(v)beta(3) could be a promising target for new agents developed for activity against this pathogen.

Involvement of the RNAse L gene in prostate cancer.

Prostate cancer is one of the most common cancers among men and has long been recognized to occur in familial clusters. Identification of genetic susceptibility loci for prostate cancer has however been extremely difficult, and only in 1996 was the first prostate cancer susceptibility locus HPC1 mapped to chromosome 1q24-25. Since, several additional putative loci have been identified by genetic linkage analysis on chromosome 1, 17, 20 and X (reviewed in). For three of these loci, family-based studies have identified three genes associated with inherited prostate cancer: the 3' processing endoribonuclease ELAC2/HPC2 gene, the macrophage scavenger receptor 1 gene (MSR1), and the endoribonuclease RNase L gene (RNAse L/HPC1). Here we will focus our review on the RNAse L gene and its involvement in prostate cancer and other diseases.

Thyroid cancer in Luxembourg: a national population-based data report (1983-1999).

BACKGROUND: Twenty years after the nuclear accident in Chernobyl (Eastern Europe), there is still a controversial debate concerning a possible effect of the radioactive iodines, especially I-131, on the increase of thyroid carcinomas (TCs) in Western Europe. Time trends in incidence rates of TC in Luxembourg in comparison with other European countries and its descriptive epidemiology were investigated. METHODS: The population-based data of the national Morphologic Tumour Registry collecting new thyroid cancers diagnosed between 1983 and 1999 at a nation-wide level in the central division of pathology were reviewed and focused on incidence rates of TC. Data from 1990 to 1999 were used to evaluate the distribution by gender, age, histological type, tumour size and the outcome. RESULTS: Out of 310 new thyroid carcinomas diagnosed between 1990 and 1999, 304 differentiated carcinomas (A: 80% papillary; B: 14.5% follicular; C: 3.5% medullary) and 6 anaplastic/undifferentiated TCs (D: 2%) were evaluated. The M/F-ratio was 1:3.2, the mean age 48.3 years (range: 13-92). The overall age-standardized (world population) incidence rates over the two 5-year periods 1990-1994 and 1995-1999 increased from 7.4 per 100,000 to 10.1 per 100,000 in females, from 2.3 per 100,000 to 3.6 per 100,000 in males. Only 3 patients were children or adolescents (1%), the majority of the patients (50%) were between 45 and 69 years of age. The percentage of microcarcinomas (<1 cm) was A: 46.4%, (115/248); B: 13.3%, (6/45); C: 27.3%, (3/11). The unexpected increase of TCs in 1997 was mainly due to the rise in the number of microcarcinomas. The observed 5-year survival rates for both genders were A: 96.0+/-2%; B: 88.9%; C: 90.9%; D: 0%. Prognosis was good in younger patients, worse in males and elderly, and extremely poor for undifferentiated TCs. CONCLUSION: The increasing incidence rates of TC, especially of the papillary type, seem mainly due to a rise in diagnosed microcarcinomas due to some extent to a change in histologic criteria and to more efficient diagnostic tools. This rise appears to be independent of the number of surgical treatments, the immigration rate, and the Chernobyl fallout as the incidence of TC in children remained stable.

RGD, the Rho'd to cell spreading.

Some RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding. However, the precise involvement of each of these recognition sites during cell adhesion is still unclear. Here we review recent investigations on integrin alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen providing evidence that the fibrinogen synergy gamma(400-411) sequence by itself promotes cell attachment by initiating alphaIIbbeta3 clustering and recruitment of intracellular proteins to focal complexes, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to induce a conformational change necessary for RhoA activation and full cell spreading.